CA1193089A - Water soluble fluors - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Water soluble fluors useful in enhancing images obtained in autoradiography have the general formula:

Description

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This invention relates to water soluble fluors. More specifically, it relates to a new class of water soluble fluors, their preparation and their use as enhancers for radioactivity detection in auto-radiography.
Auto-radiography is the production of an image in a photo-graphic emu1sion by a radioactively labelled substance. Auto-radiography is an important method in biological, biochemical, and clinical investigations and analyses. One of its principal uses is to locate bands of radio labelled materials, e.g., rnolecules radio 11 d ith 3H 14C 32p 35S or 125I on chromatographY plates or on slab gels used for electrophoresis. The usefulness of auto-radiography, however, has freque~t;y been limited because of the long exposure times usually required for the low radiation levels of isotopes incorporated into the plates and gels and, in some cases, the very weak energies of radiation.
One approach taken to overcome that problem has been the use of scintillation materials which act as amplifiers of the exposure process. Radiation energy causes the fluor present in the system to emit light on a certain wavelength, and the light exposes the photo-graphic emulsion in a way that is much faster and more efficient thancould be obtained by relying on auto-radiography alone. The combina-tion of auto-radiography and fluorescence is called Fluorography or autofluorography. Bands of material labelled with radioactive isotopes can be detected more readily and rapidly in, for example, slabs of electrophoresis gel, by means of fluorography.
In chromatography and electrophoresis, the radioactive material to be measured is absorbed or adsorbed according to conventional techniques on or in an organic or inorganic absorbent or adsorbent layer or column of separation medium or material, e.g. silica gel, alumina, cellulose, polyamide, polyacrylamide, cross-linked dextran~ agarose, etc., which is usually support;ed on a plate, e.g. glass or plastic sheet. This is called a chromatogram or electrophoretogram.

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Two of the most common separation media used in electrophoresis are aqueous polyacrylamide and agarose yels.
Gel electrophoresis is a method of separating charged parti-cles, such as proteins, whereby the charged particles move through a gel medium under the influence of an applied electric field, their rate of movement through the lattice formed by the hydrated gel being depen-dent on charge and molecular size or weight. When the electric field is removed, the particles are present in the gel in discrete bands which can either be sliced up for liquid scintillation counting, or in the case of radionuclides such as tritium which emit lower energy particles, more preferably analyzed by fluorography.
In the case of radioactive labelled animal tissue.s, e.g.
tissue auto-radiography, the radioactive material is usually adrninis-tered to the live animal and becomes selectively absorbed or adsorbed into certain tissuesSo that the tissue, usually in the form of a thin slice, may be considered as the absorbent or adsorbent layer. In the case of paper chromatography the paper (cellulose) is the adsorbent.
Where the adsorbent material is in the form of a thin layer supported on a plate, it is called thin layer chromatography and a thin layer chromatogram.
In auto-radiography, the radioactivity of the material being tested is measured by a film sensitive to radioactivity.
In autofluorography, a fluor or scintillator, which is excited or stimulated by radioactivity to emit light, is applied in close proximity to the radioactive material and the intensity of light emission is measured by a photographic film, which is sensitive to light. Conventionally, the photograph is taken with the radioactive sample sandwiched against the emulsion of the film.
Fluorography has important advantages over conventional auto-radiography5 the most important of which is a markedly shorter exposure time (typically shortened from two weeks to 16 to 24 hours) with wea~

2 -radioactive emitters, such as tritium.
However, in spite of this important advantage, presently known autofluorographic techniques have serious disadvantages, parti-cularly in systems where relatively thick layers of absorbent or adsorbent materials are used in the separation process, e.g., poly-acrylamide gel electrophoresis which is frequently used in receptor site, nucleic acid,and enzyme research.
One of the problems is in developing a method for placing and maintaining the scintillator fluor in close proximity to the radioactive emitter. If not in close proximity, a portion of the emitted radioactive particles will not reach the scintillation fluor.
In the case of thin layer chromatography, the scintillator fluor has been dissolved in a suitable carrier, e~g. benzene or toluene, and then sprayed onto the thin layer separation medium, e.g. a paper strip, containing the radioactive sample. After drying, a piece of film sensitive to the light emission of the scintillator may then be juxtaposed and this sandwich is allowed to stand for a time sufficient to achieve exposure. In such a system, it is difficult to evenly distribute the scintil1ator fluor, the radioactive material may spread and diffuse, and the small crystals of scintillator fluor tend to be so loosely bound that great care must be exercised in handling the sample.
In addition to the above disadvantages it -is sometimes desirable to use thicker layers of adsorbent or absorbent material.
Once any appreciable thickness is used, i.e. greater than about 0.1 mm, the technique of spraying no longer places the scintillator fluor in close enough proximity to enough of the radioactive material. This results in a drastic loss in the ability of the scintillator fluor to be excited by the emitted particles and convert them into light.
Accordingly, it is necessary to somehow transport the scintillator fluor into the interior of the separation medium. One method for accomplishing this transportation is by soaking the ~3~
separation medium of absorbent or adsorbent material in a bath contain-ing the scintillation fluor dissolved in a suitable carrier.
A number of fluorography systems are currently available.
One system was described by eonner et al, Eur. J. Biochem. 46:No. 1 83-88 (197~).
In that method the radioactively-label1ed protein is separated by electrophoresis using an aqueous polyacrylamide gelg followed by soaking the gel in about 20 times its volume of dimethyl-sulfoxide (DMSO) for 30 minutes, and then immersed a second time for 30 minutes in fresh DMSO to displace all the water from the gel. The next step is to soak the gel in a 20% (w/w) solution of 2,5-diphenyl-oxazole (PPO) in DMSO to impregnate the gel with scintillator which is th~n precipitated in the gel by washing with water. The gel is finally dried and exposed to the film. This technique has numerous disadvan-tages, many of which are reported in the appendix of an article by Laskey and Mills in the Eur. J. Biochem., Vol. 56, pages 335-341 (1975).
Agarose gells containing less than 2% polyacrylamide (plus 0.5% agarose) or agarose alone dissolve in DMSO unless methanol is substituted for the DMSO. Even this substitution is only effective for gels having less than 2% polyacrylamide, since gels having higher concentrations cf polyacrylamide shrink severely when contacted with methanol. Even at 30% methanol, shrinkage of higher polyacrylamide concentration gels may take place. Another disadvantage is that the failure to remove all the DMSO may result in adhesion of the film to the gel and artifactual blackening of the film. Another disadvantage is the ability of DMSO
to penetrate through the skin of anyone handling it by itself or the gel which has been soaked in it, thereby carrying toxic dissolved material with it through the skin as well as imparting a garlic smell to the person's breath. Another disadvantage is that the gels must be soaked in the DMSO-fluor solution for as much as 3 hours to obtain complete impregnation. A further disadvantage is that high concentra-tions of PPO, concentrations between 14% and 19% (w/w) being typical, must be used in the impregnation solution. Another disadvantage with DMS0 as well as with other conventional carriers is that whi1e PPO is efficient in converting absorbed radiation into photons of light, it is somewhat 1imited in its abi1ity to absorb the energy emitted by the radioactive emitter. Another disadvantage is that the soakings in DMS0 to dehydrate the ge1 are time consuming.
One method for increasing the absorption ability of PP0 when thin layer chromatograph is being employed is described in Bonner and Stedman, Analytical Biochemistry, Vol. 89, pages 247-25~ (1978). Three methods for detection of 3H and 14C in silica gel thin layer chromato-grams are described in that article. The first method utilizes 2-methylnaphthalene (2MN) which i5 described as being a scintil1ation so1vent for use in solid systems by ana10gy to scinti11ation f1uids which many times contain a solvent in addition to the scinti11ator.
As in liquid systems, the solvent molecules collect the energy from the emitted beta radiation and transfer it to PP0 molecu1es, which then emit photons of light. A solvent is a compound which converts the kinetic energy radiated by the radioactive emitter to electronic excitation energy and transfers that energy to the fluor dissolved therein. The first method comprises dipping the dried thin layer p1ates in a solution of 2MN which has been liquified by heating and which contains 0.4% (w/v) of PP0, unti1 they are soaked and then re-moving the p1ates from the solution. When the so1ution has so1idified, the plate is placed against film and exposed. An alternative, ir spraying is deemed to be more desirable, is to replace 10% oF the 2MN
with toluene to make the solution a 1iquid at room temperature. The second method invo1ves dipping the p1ates in an ether so1ution con-taining between 7% and 30% (w/v) of PP0, drying the p1ates and then exposing as above,with better sensitivity being seen as the PP0 con-centration increases. The third method involves dipping the thin layerp1ates in me1ted PP0 until soaked, removing and then heating unti1 the excess PP0 has drained off, and exposing to fi1m as above. While ,,i i~
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useful in thin layer chromatography, numerous problems exist in attempting to use such systems with other media. One problem is that neither PPO nor 2MN is soluble nor miscible in water to any appreciable extent. Accordingly, aqueous polyacrylamide or agarose gels are not impregnated with PPO nor 2MN while in the hydrated state, nor evenif dried since the lattice structure ccllapses upon drying.
Secondly, PPO and 2MN are very expensive even if it were possible to use themin such systems. The second method also is not useful with aqueous gels since ether and similar solvents such as alcohols cause drastic shrinkage of such gels. Furthermore, relatively high (7% to 30%) concentrations of expensive PPO in the ether are required for efficient fluorography.
Another fluorographic system has been described in U.S.
Patent No. 4,293,436, issued October 6, 1981 to Dennis L. Fost. In that method, the aqueous separation medium or other medium to be sub-jected to fluoro~raphy is impregnated with a water-soluble or water-miscible lower alkyl carboxylic acid in which a scintillator fluor has been dissolved or dispersed, followed by precipitation of the fluor within the gel or tissue by aqueous soaking. However, that procedure also suffers from disadvantages. Handling of the fluors, which are generally soluble only in organic solvents, requires impregnation times in subsequent manipulation steps that are long as compared with the times of the present invention, e.g., two to three times the periods required with this invention. The fluoroscent system may also fade with time, and its enhancement drop within a relatively short period of time. Further, the treatment with highly concentrated carboxylic acid~and the further treatment of extended aqueous soal~ing, both tend to adversely affect the sharpness of the bands, thus decreasing the accuracy of the procedure. The carboxylic acids may also cause the gel being treated to swell, and this requires the addition of an anti-swelling agent.

Another system, not necessarily prior art to the present invention, i5 now being marketed by National Diagnostics under the trade mark AUTOFLUOR. The exact nature of that material is not known, but a sample obtained some months ago appeared to contain a naphthol disulfonic acid. The material was unstable, and thus could not be utilized effectively after relatively short periods of time. A more recent sample seems to be based on sodium salicylate. For the dis-advantages of using sodium salicylate, see J. P. Chamberlain, Anal.
Biochem. 9~:132-5 (1979).
It is an object of this invention to provide a new auto-fluorographic enhancer, containing water soluble fluors which elimi-nate the problems associated with the impregnation of gels, and permit wider and more convenient use of fluorography.
Ott et al, J._Am Chem Soc., 7J3:1941 (1956) reported the preparation of 2,5--diphenyl-3-methyloxazolium salts, which were apparently soluble to some degree in water and had some fluor proper-ties. However, these compounds are only stable in acidic solutions, and are rapidly and quantitatively converted to the N-methyl-alpha-acylamido ketone by hydrolytic ring cleavage in alkali. Accordingly, their use as auto-radiographic enhancers is severely limited.
Bodendo et al, Archiv. der Phanm., 298:Y93 (1965) reported the preparation of 4-[(2,5-diphenyloxazolyl) methyl] piperidinium hydrochloride and 4-~(2,5-diphenyloxozolyl) methyl] morpholine. How-ever, those materials were neither synthesized, ~ormulated nor tested for use in fluorography.
Certain water soluble compounds, such as alpha-naphthol polyethylene glycol (Naftaxol*-Hoechst), p-octylphenolpolyethylene glycol (Triton*-X-100, Rohm & ~laas) and p-nonylphenylpolyethylene glycol (Igepal*-C0730, GAF) are known surfactants. Although those compounds do in fact posess some fluorescent properties, they have not been utilized as enhancers for use in auto radiography. Typi-cally such materials have low quantum e~ficiencies, e.g., below 0.

*Trade Mark to well below 0.1, and many such mat~rials may fluoresce at wavelengths which are not suitable for fluorography The present invention contemplates a new system of fluoro-graphy, using certain water soluble fluorescent materials, both neutral and charged. Most of the fluorescent materials described herein have been prepared for the first time. They are stable at elevated tempera-tures, and their water solubility, thermal stability9 and immobility in gel assures their effectiveness. The materials described are highly effective in enhancing the image obtained in fluorography, when used either singly or as matched pairs or sets of enhancers. Further, the systems used in incorporating these enhancers are stable for long periods of time, both in the bottle and in ~he gel, which can be impor-tant for long exposure testing or in the common situation in which it is desired to store the gel and retest it at a later date (e.g., use as a standard).
The enhancer compositions of the present invention generally contain a fluorescent material having the following formula:
[F ~ _ ~ S]z (1) wherein F is a moiety which absorbs energy and emits electromagnetic energy and thus acts as a fluor, 5 is a hydrophilic surfactant moiety which renders the molecule water soluble, and B is a structure which binds the surfactant to the fluor. The value subscripts x, y and z may be between 1 and 1~ and are preferably between 1 and 3. Where x, y or z is more than one, each F, B or S may be the same or may be different.
Thus, in accordance with one aspect of the invention, there is provided a method of enhancing the production of auto-radiographic images by radioactive emitters contained in an absorbent or adsorbent separation medium which comprises contacting the separation medium with a fluorographic enhancer composition which contains ~ e or more fluorescent materials as described above.
In accordance with another aspect of the invention there is !~ ~
~ , - 8 provided a composition of matter comprising at least two fluorescent materials as described above.
In aceordance with still ano~her aspect of the invention, there is proYided a composition of matter comprislng a fluorescent material as described above, having a quantum e~ficiency above about 0.6, and an acceptable carrier therefor. In particular embodiments of the invention an article of manufacture is provided comprising an absorbent or adsorbent separation medium containing this composition, in particular the separation medium may be an electrophoresis gel.
In a further particular embodiment, the article further comprises a layer of photographic film attached to said separation medium, which film is sensitive to electromagnetic energy at a wavelength corresponding to the emission wavelength of at least one fluor contained in the separation medium.
The fluor portion of the molecule may be derived from any of the known f1uors which efficiently collect the radiation from the radioactive labelled compound and emit light at a wavelength corres-ponding to the sensitivity of the photo emulsion, and which is chemi-cally inert to the other components of the fluorographic composition and the gel. Preferred fluors are those which are stable and have a high quantum efficiency, i.e., those that are efficient in converting received radiation into light. Preferably the fluors in accordance with the present invention have a qulntum efficiency of at least above about 0.1, more preferably above about 0.2, and most preferably at least one f1uor in the fluorographic enhancer composition of the pre-sent invention has a quantum efficiency above about 0.6. Preferably the F portion of the mo1ecule of equation (1) is a radical derived from substituted or unsubstituted 2,5-diphenyloxazole (PP0), e.g., 2,5-diphenyl-4-methyloxazole~ naphthalene and its derivatives, e.g., 1-methyl naphthalene, unsubstituted or substituted terphenyl, e.g., m-terpheny1, p-terphenyl, 3,3'-dimethyl-p-terphenyl, substituted or unsubstituted fluorene, e.g., 1,2-benzofluorene or l-methylfluorene, _ g _ isopropyl phenyl biphenylyloxadiazole (isopropyl PBD), 2-[1-naphthylJ-5-phenyloxazole (B-NP0), t-butyl phenyl biphenylyloxadiazole (butyl PBD), p-quaterphenyl, or derivatives of other fluors known per se in - 9a -~43~
the art. See, e.g., Berlman, ~andbook of Fluorescence Spectra o~
Aromatic Molecules (Academic Press1 N.Y., 1971~. Presently preferred are derivatives of PPO, naphthalene, p-terphenyl and fluorene. Highly advantayeous results can be obtained with fluorescent materials (compounds or compositions) containing derivatives of a plurality of fluors, such as combinations of derivatives of PPO and naphthaleneS of terphenyl and naphthalene, of PPO and fluorene, etc.
The surfactant S part of the molecule of formula (1) may be any surfactant which can be bound to the fluors described above to make the resulting molecule water soluble or water miscible, stable and non-reactive with the other components of the system. The surfactant used may be anionic,cationic, non-ionic, or amphoteric. They may be known surfactants, such as polyethylene glycol derivatives, aryl sulfonates, amines, quaternary ammonium salts, or, they may be pro-surfactants, which, as used herein, shall mean moieties which, when chemically combined with the ~luor in accordance with formula (1), make the resulting compound water soluble or water miscible. A preferred example of the latter is the sulfonic acid radical, or salt thereof, which when combined with aryl fluors, can result in arylsulfonates which have hydrophilic surfactant properties. Presently preferred examples of S in formula (1) are sulfonic acid or salt residues (-S03H or -S03); polyethylene glycol, preferably having a molecular weight of about 100 to 10,000, more preferably 200 to 1500, most pre-~erably about 300 to 1200; substituted or unsubstituted aryl or alkaryl sulfonates, such as benzene sulfonate, toluene sulfonate; amines and derivatives thereof, such as methylamine; amides, e.g., acrylamide;
imides such as phthalimide and residues which, together with [F]x or tF~X and [B]y in formula (1) create quaternary ammonium salts, e.g., trimethyl ammonium chloride, N-benzyl,-N,N-dimethyl ammonium chloride.
Other suitable reactable surfactants or pre--surfactants are known or readily apparent to those oF ordinary skill in the art. See, e.g., McCutcheon's Publications/1980 (MC Publishing Co., Glen Park, NJ (1980))~

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The binder B in formula (1) may be any moiety which chemi-cally bonds the fluor with the surface active agent, without destroy-ing the properties of either, and which, when in combination ~t/ith the fluor and the surfactant, is not reactive with the other necessary components of the system. B may simply be a chemical bond, or may be an alkylene linkage, pre-ferably a substituted or unsubstituted lower alkylene linkage of 1-6 carbon atoms, e.g., methylene, ethylene, propylene, etc.; alkenyl moieties, such as ethenylene, propenylene, etc.; carboxy moieties such as carbonyldioxy, malonyl, methylenedioxy, epoxy, etc.; arylene, e.g., phenylene, diphenylene, etc.; alkarylene, e.g., methylphenylene or styrene; or it may be thio or another sulfur group.
The compositions of the present invention can be obtained from commercially available products, by reactions which the skilled in the art will readily appreciate in the light of the present dis-closure. For example,fluor compounds having, or modified to have, alkyl halide substituents may be reacted with nucleophilic substituted surfactants or pre-surfactants having a wide variety of reactive groups, including hydroxy, alkoxy, carboxy, amino, thio and other groups. Thus fluors having alkyl halide groups can be reacted with surfactants or pre-surfactants having available hydroxyl groups, to form ether linkages (e.g., polyethylene glycol surfactants of var-ious chain lengths) ethoxylated alcohols, fatty alcohols and derivatives thereof etc.; with surfactants or pre-surfactants having available carboxyl groups to form ester linkages, (e.g., Fatty acids and derivatives), with surfactants having available substituted or un-substituted amino groups to form substituted amino linkages, (e.g., ammonia or primary, secondary or tertiary amines), etc. Where the fluor contains an aryl group or other group subject to s~lfonation, the fluor can be sulfonated to yield water soluble aryl sulfonates in known manner (e.g., sulfonation of terphenyl in fuming sulfuric acid to yield terphenyl sulfonates). Other reactions between fluors and , .

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surface active agents having different reactive groups wi11 be readily apparent to those skilled in the art from this disclosure.
The scin-tillator compositions of tne present invention can advantageously contain a number of other materials. In addition to a solubilized primary fluor, the scintillator composition can contain a secondary fluor, or spectrum shifter, which absorbs light at the wave-lengths emitted by the primary fluor and emits electromagnetic energy at a wavelength to which the photographic film is sensitive. The secondary fluor can be water solublized by reacting with a surface active agent and as linking structures in the same way the primary fluor is solubilized as described herein. A wide variety of secondary fluors are available, including p-bis-[2-(4-methyl-5-phenyloxazolyl)]
benzene (called dimethyl POPOP), or p-bis-(o-methylstyryl) benzene (called bis-MSB), p-p'diphenyl stilbene, 9,10-diphenyl anthracene, POPOP, and 2,5-diphenyloxazole (PP0). Suitable matched combinations of materials include solubilized PP0 and solubilized POPOP, or solubi-lized PP0 and solubilized bis-MSB, etc. Preferred combinations of fluors for use in fluorography, which achieve good fluorescence in the range of sensitivity of commercial x-ray film, include solubilized PP0 or suitable derivatives thereof as secondary fluors and solubilized naphthalene, anthracene, terphenyl, etc. Preferably, the enhancing compositions contain at least one -Fluor or clerivatives thereof having a quantum efficiency of about 0.2, more preferably above about 0.~ or 0.6 in the composition. Preferably the quantum efficiencies of all fluors in the system are high9 e.g., above about 0.25, more preferably above about 0.3, although systems which contain a mixture of fluors of varying quantum efficiencies, e.g., a mixture of solubilized naphthalene having a relatively low quantum efficiency (e.g.9 naphthalene sulfonate) and solubilized PP0 (e.g., PP0 sulfonates) have been found to be very effective in enhancing fluorographic exposures.
The scintillation compositions of the present invention can a1so advantageously incllJde an antioxidant to prevent decomposition of the fluor or other components during storage. A wide variety of anti-oxidant materials are kno~ln to the art and any of these is acceptable, provided that they are inert to the other ingredients, to the gel or chromatrographic material, and to the composition being analyzed.
Examples of antioxidants for use with the present invention would include ascorbic acid, citric acid and butylated hydroxytoluene (BHT).
Still other materials may also be included. See, e.g., U.S. Patent No. 3,068,178.
Although the materials and methods of the Fost patent, No.
4,293,436, were a major advance over the previous1y used method of Bonner et al, the Fost materials still suffered from substantial disadvantages. The fost compositions required a first step of soaking the gels for one hour or more in the Fost composition to impregnate the scintillation composition into the gel structure, and followed by immersion in water under agitation for one hour or more in order to precipitate the Fost water-soluble materials within the gel, followed by drying and exposure to the photographic film. The immersion in water tended to blur or displace the lines of materials in the gel, and added substantial time to the Fost procedure. The Fost composition also contained noxious compounds which were unpleasant to deal with.
Largely because of their water soluble nature, the scintilla-tion compositions and fluors of the present invention are much simpler, safer, and more pleasant to use, and save very substantial amounts of time as compared to the compositions and methods of either Fost or Bonner et al. The fluors of the present invention are generally water soluble and inert. Those of the present fluors which are either nonionic or can be copolymerized with the separation material can be simply incorporated in the electrophoresis gel, chromatographic media, etc. before the electrophoresis or chromatography is run. This procedure requires no special impregnation or other procedure after electrophoresis. For example, an electrophoresis gel slab which already contains the fluor need only be dried in the normal manner and exposed ~3~8~

to the photographic film. The resulting exposures are clearly enhanced by the presence of -these fluors.
However, it is not necessary to incorporate the fluors in the gel or other material itself before electrophoresis in order to obtain the benefits oF the present invention. Standard gels need only be soaked, in the scintillation compositions of the present invention for a much shorter period of time than the Fost compositions, to allow the fluors to be impregnated in the gel. Preferably the gel ~s soaked from 5 to 45 minutes, more preferably from about 10 to 20 minutes and the resulting gel after electrophoresis, for example, need only be dried and exposed to photographic film. Because the scintillation composi-tions are water soluble, they are readily impregnated into the gel, and no dehydration step is required in the method of Bonner et al and no precipitation step as required by Fost is necessary. Thereforeg the time between the completion of electrophoresis or chromatography and the start of film exposure is substantially reduced.
When utilized as part of the electrophoresis gel or chromato-graphic material, the fluors of the present invention, either alone or in combination with each other or with other materials, may be present in at widely varying ranges of concentration. Generally the fluors should be nonionic in nature, so as not to migrate under the electric field. The fluors should be present in at least sufficient quantities to provide enhancement of the photographic exposure, but not so con-centrated as to adversely affect the ability of the gel or other material to polymerize, the motility of the sample components through the electrophoresis gel, or the sorbtion characteristics of the chromatographic material. Broadly the fluor~s) may be present in from about 1% to about 100% by weight based on the weight of the acrylamide or other monomer, i.e., based on the weight of the solids content of the separation medium; preferably from about 5% to about 85% by weight;
most preferably from about 20 to about 30% by weight.

More than one water soluble fluor can be polymerized or other-,,, ~
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3~8~3 wise incorporated into the electrophoresis or other gel material. For example, copolymers of acrylamide monomers with two or more water-soluble fluors of the present invention are particularly advantageous embodiments of the present invention. The amounts of water soluble fluors in the copolymer may vary from that amount which just gives enhancement to essentially purely comonomers of those fluors, but pre-ferably the fluors are about 5-~5% of the monomer. Preferably the weight ratio of water soluble fluors to monomer is within about 23~, to about 76%, most preferably 20 to 30% by weight of the copolymer.
The molecular ratio of one water soluble fluor to another in the co-polymer may broadly range from 100:1 to 1:100, but preferably ranges from about 15:1 to 1:15. That ratio will depend to some extent on whether one of the water soluble fluor monomers in the copolymer is serving as a secondary fluor for the other water soluble f1uor(s) in the copolymerS as will be appreciated by those skilled in the art from this disclosure.
Where the fluor is to be incorporated into the gel or other material by soaking the material in a solution of the fluor(s) in an impregnation step, the ranges of concentrations useable are similarly broad. Generally the fluor(s) may be present in amounts ranging from about 0.001 molar to about 2.0 molar; preferably about 0.005M to about l.OM, more preferably from about 0.15M to about 0.45M. Generally, the higher the concentration of fluor(s) in the impreynation bath, the faster the fluor(s) will diffuse into the gel or chromatographic material, but increasing viscosity, increased difficulty of handling, and adversely affected rnass transfer coefficients may be encountered at higher concentrations.
The present invention will be further understood with refer-ence to the following illustrative embodiments, which are exemplary only, and not to be taken as limiting the invention.

- ~5 -1~3~9 Preparation of 4-chloromethyl-2,5-diphenyloxazole.
100 grams (613 millimoles (mM))of Isonitrosopropiophenone (available from Eastman Organic Che~icals) and about 65 grams (613mM) of benzaldehyde are dissolved in glacial acetic acid and ~IC1 gas was bubbled through the solution with stirring until a yellow precipitate was found. The precipitate was collected and washed with ether until it was white. This product was dissolved in methanol with some heating, and neutralized with sodium hydroxide. The product had the formula \ N
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This product was dissolved in ethanol, placed in a sealed reaction container with freshly activated Raney-nickel catalyst and degassed by vacuum. The systems were then charged to a pressure of about 3 atmospheres with hydrogen gas. The reaction was continued, with supplemental hydrogen being added, until hydrogen was no longer consumed; and thin layer chromatography using 8:1 hexand/ethyl acetate on silica gel showed no starting material. This took a period of approximately 2 hours. Then the catalyst was filtered, the solution distilled off, and the resulting white crystals of 4-methyl-2,5-di-phenyloxazole were dried in a vacuum oven~
Five grams of this product (about 21 mmol) was dissolved in carbon tetrachloride. A catalytic amount (about 25 mg) of benzoyl peroxide was added, and the solution was heated to reflux. About 1.7 ml (about 21 mmol) of sulfuryl chloride was added dropwise to the re-fluxing mixture and refluxing was continued for about an hour. There-after the mixture was allowed to cool to room temperature, where it stayed overnight. The solvent was removed under reduced pressure, and the remaining product, 4-chloromethyl-2,5-diphenyloxazole, was ~3~

recrystallized from e-thanol. Yield was 3.8g (67%), rnelting point 13)3-9~C.
~XAMPLE 2 Preparation of Methoxypolyethyleneglycol 4-(2,5-diphenyloxazolyl) methyl ether.
About 7.5 grams (10 mM) of a polyethylene glycol monomethyl ether having an average molecu'ar weight of about 750 was dissolved in 75 cc of dry toluene. The polyethylene glycol monomethyl ethers are readily available in the molecular weight ranges discussed herein (e.g.
from the Aldrich Chemical Company), and/or are easily prepared by those skilled in the art. A small portion of toluene was distilled to ensure that the reagent and the solvent were completely dry. The toluene solution was cooled to room temperature, and 0.3 g (12.5 mM) of sodium hydride was slowly added. The mixture was then stirred under argon at room temperature until no further hydrogen was evolved. About 2.5 9 (9.5 mM) of 4-chloromethyl-2,5-diphenyloxazole, dissolved in about 20 cc of toluene, was added to the resulting mixture. The mixture was the refluxed until all of the 4-chloromethyl-2,5-diphenyloxazole had reacted. The reaction was monitored by thin layer chromatography utilizing a solvent consisting of 1 volume ethyl acetate to 8 volumes of hexane, on a silica gel plate. The salt was separated by filtra-tion, and the product was treated with decolorizing charcoal, and sub-jected to solvent removal in a vacuum. The resulting product was obtained in the form of an oily residue, which solidified on standing at room temperature.
EXAMP E_ Preparation ~
Polyethylene glycol having an average molecular weight of about 400 was dissolved in about 75 cc of toluene. A small portion of toluene was distilled to ensure that both the reagent and the solvent were complete dry. To the cooled solution, about 1.1 9 (45 millimoles) of sodium hydride was added. The mixture was stirred under argon at room temperature until no further hydrogen was evolved.
7 9 (40 mmoles) of l-(chloromethyl) naphthalene was added~ and the mixture was refluxed until all of the 1-(chloromethyl) naphthalene had reacted. The l-(chloromethyl) naphthalene is commercially available, e.g., from Eastman Organic Chemicals Division of Eastman Kodak. The reaction was monitored by thin layer chromatography using the ethyl acetate/hexane systems of Example 2. The reaction mixture was treated with decolorizing charcoal, yielding the product after vacuurn filtra-tion. The product was oily and not soluble in water.

Preparation of Polyethyleneglycol di-l-Naphthyl Methyl Ether.
By the same method, polyethyleneglycol di-1-naphthylmethyl ether was prepared from a polyethyleneglycol having an average mole-cular weight of about 600. The product was oily and partially soluble in water.

of Polyethylene glycol di-1-Napthyl Methyl Ether Polyethyleneglycol having an average molecular weight of about 1,000, (10 9; about 10 mM) was dissolved in 75 cc of toluene. A small portion of toluene was distilled until no further azeotrope came off.
To the cooled toluene solution was added 0.6 9 (25-moles) sodium hydride.
The mixture was stirred under argon at room temperature until no further hydrogen was evolved. To this mixture 3.4 9 (l9-millimoles) of 1-(chloromethyl) naphthalene was added. The mixture was refluxed until all l-(chloromethyl) naphthalene had reacted. The reaction was monitored by the above described thin layer chromatography. The re-action mixture was treated with decolorizing charcoal, and the product was obtained after removal of charcoal by vacuum filtration and solvent distilled under vacuum.
_XAMPLE 6 Preparation of 4-[5-~2-phenyloxazolyl)] Benzene Sulfonic Acid.

66.7 g of PPO (0.3 moles) was slowly added to 100 ml of fuming 3~

sulfuric acid with stirring. When the exothermic reaction subsided, the reaction mixture was stirred for an additional 1/2 hour, then poured into approximately 200 ~ of cracked ice. A white precipitate was formed immediately. This was collected, washed several times with ice cold water and finally with methanol.
Yield 80 g (90,~) M.P. > 329C.
The product WdS then recrystallized from a mixture o-f methanol and water havin~ 5 parts by weight methanol to 7 parts water.

C H N
Analysis for Calculated 56.42 4.i 4.3 Monohydrate Found 57.16 4.09 4.31 Hereafter that product is called "PPO-S03H".

Preparation of Terphenyltrisulfonic Acid Trisodium Salt.
__ ___ 46.5 (0.2 moles) of p-terphenyl was added slowly to 100 ml of fuming sulfuric acid, with stirring. When the exothermic reaction subsided, the reaction mixture was heated at approxirnately 100C for one hour, then poured into approximately 200 9 of cracked ice. Sus-pended solid was filtered off, and sodium chloride was added to the filtrate until saturation.
A white precipitate, the trisodium salt of terphenyltri-sulfonic acid, was filtered, washed with ice water, and finally washed with methanol.

C H
Analysis Calculated 40.23 2.25 Found 40.67 2.21 EXAMPLE ~
Preparation of Fluorene-2,7-Disulfonic Acid Disodium Salt.
33.3 g of fluorene (0.2 moles) was stirred with 75 rnl of 98%
H2S04 at 80-90C for 15-20 minutes, at which time the reaction mixture solidified. The greenish, pasty mass was cooled and added to 250 ml of cracked ice. The solution was made basic with 50~ NaOH solution, and the resulting product, a white precipitate, was collected and recrystallized from water. Yield 75~.

33(~

Analysis Calculated C 38.43 ~ 2.98 Found C 37.~2 H 2.51 The structure was also confirmed by NMR analysis, showing peaks at 3.50 (2H) and 7.5-8.00 (6H).

-Preparation of 2,5-Diphenyl-3-Methyloxazolium Toluenesulfonate.
10 9 of 2,5-Diphenyloxazole was heated at 125C for 5-10 minutes with 30 9 of methyl toluenesulfonate, both commercially available compounds. The solution was cooled, and anhydrous ether was added to precipitate the product. The product was recrystalli~ed by dissolving in absolute ethanol and then adding ethyl acetate. The product was obtained in 95% yield, and had a melting point of 167-168C.

Preparation of 4-Phthalimido Methyl-2,5-Diphenyloxazole.
.
A solution of potassium phthalimide (10 9, 54 mmoles) and

4-chloromethyl-2,5-diphenyloxazole (10.4 9, 40 mmoles) in 80 cc of dimethylformamide was heated at 100C for 2 hours. When the solution was cooled to room temperature 300 cc of water was added. The solu-tion was extracted with chloroform several titnes. The combined chloroform solution was washed with 20-30 ml of 0.2N sodium hydroxide and finally with water. After drying over sodium sulfate the chloro-form was concentrated. The product precipitated out.
Yield 11 9 (73%) M.P. 192-3C
C H N
Elemental Calculated 76.0 4.22 7.37 analysis Found 76.10 4.20 7.50 _ Preparatlon o-f 4-Aminomethyl-2,5-Dip_ n _ xa~ole.
A mixture made up of 20 ml of 48~ hydrobromic acid, 20 ml of glacial acetic acid and 10 9 of 4-phthalimidomethyl-2,5-diphenyloxa~ole, was heated under reflux until a clear solution resulted (3 hrs.). The solvent was pulled off under vacuum until dryness. The residue wa~

A, ~
~ - 20 ~

dissolved in lOO ml of water, made basic with lN NaOH and extracted with ethyl acetate. The ethyl acetate solution was washed with water until neutral and dried over sodium sulfate. The solvent was pulled oFf under vacuum and the residue was recrystallized from ethyl acetate and hexane.
Yield 5.0 9 (90%) M.P. 103-4C.

C H N
Analysis Calculated 76.80 4.80 5.60 Found 75.64 4.97 5.43 lOEXAMPLE 12 Preparation of N-[4-(2,5-Diphenyloxazolyl) methyl] Acrylamide 1.3 cc (15 g, 16 mmoles) of acryloyl chloride in 5 cc of ethyl acetate was added dropwise to a solution made up of 3 g of 4-aminomethyl-2,5-diphenyloxazole (12 mmoles), 15 cc pyridine and 50 cc of ethyl acetate. When the reaction was complete, the product was washed with water and dried over sodium sulfate. The solvent was pulled off under vacuum. The residue was recrystallized from ethyl acetate hexane mixture.
Yield 3.14 g (90%) M.P. 169-700CA

Preparation of N-[4-(2?5-diehenyloxazolyl) methyl]-N-benzyl-N, N-dimethyl ammonium chloride.
__ . ___ A mixture made up of 4-chloromethyl-2,5-diphenyloxazole (6.2 g, 24 mmoles) benzyldimethylamine (10 g, 70 mmoles) and 60 ml of ethyl alcohol was heated under re-flux for 2 hours. The product was isolated by addition of ethyl acetate.
Yield 9.3 g (100%) M.P. 188-190C.

C H N
Elemental Calculated 76.26 6.14 6.93 analysis Found 76.64 6.35 6.75 ~ 3~

_reparation of N [4-~ _5-diphenyloxazolyl)methyl]-trimethyl mmonium chloride_ Trimethy1amine was bubbled into a solution of 4-chloromethyl-2,5-diphenyloxazole (5 9, 18 5 mmoles) in 80 cc of ethanol. An exothermic reaction ensued. When the solution was saturated with trimethylamine it was stirred for 20 minutes. The product was pre-cipitated by the addition of ethyl ether.
Yield 5.8 9 (100%) M.P. 248-250~C.

C H N
Elemental Calculated 69.51 6.40 8.54 analysis Found 68.89 6.61 8.39 Preparation of a Water Soluble Terpolymer Containing N-[4-(2,5-___ diphenyloxazolyl) methyl] acrylamide, N-[l-naphthylmethyl] acrylamide and N-Hydroxymethyl acrylamide.
The terpolymer was prepared by the free radical polymerization of the components using benzoyl peroxide as catalyst at 70-80C 30mg of N~[4-(2,5-diphenyloxazolyl) method] acrylamide~ 232mg of N-[1-naphthyl-methyl] acrylamide and 3.64 grams of a 60% aqueous solution of N-hydroxymethyl acrylamide were heated in excess ethanol under nitrogen atmosphere with agitation at about 70C. Af-ter stirring for about 10 minutes at 70C while nitrogen was bubbled through the reaction mixture, a white precipitate formed. The reaction was allowed to reflux for about 5 hours, and its progress was monitored by thin layer ehromato-graphy, using a 1 to 1 mixture of ethyl acetate and hexane on a silica gel plate. Thereafter the solvent was removed on a rotary evapora-tor.
This terpolymer may then be copolymerized with an electrophoresis gel, e.g., a 5% polyacrylamide get, and will result in enhancement of autoradiographs made using the resulting gel.

~ 22 -3~
EXAMPLE_16 Gel Electrophoresis A typical slab gel electrophoresis, acrylamide is poly-merized into a thin rectangular slab between two glass plates. Sample wells are made at one end of the gel by placing a comb-shaped form into the reaction mixture before it polymerizes. After polymerization the form is removed, leaving sample wells molded into the polyacrylamide gel.
The electrophoresis apparatus is composed of two buffer resevoirs or wells. The anodic lead from the power source is immersed in the upper buffer wel1. The cathodic lead is contained in the lower buffer well. The circuit is completed by the poly-acrylamide gel. The power source used is preset for constant voltage or constant current. In the following examples, the gel used was generally a 5% (%T) polyacrylamide gel containing about 2.7% N,N'-methylene-bisacrylamide, based on the weight of the monomer. The gel dimensions are approximately 11 cm x 12 cm x 0.15 cm. The buffer system is a Tris-borate EDTA (TBE) system. The running buffer con-tains 90 mM Tris(hydroxymethyl)aminomethane with boric acid added until pH 8.3 and containing about 2.5 mM EDTA (ethylene diamine tetraacetic acid) disodium salt.
The samples run on the gels are New England Nuclear product NET-64~*, which contains a set of 11 DNA molecular weight markers, having a molecular wei~ht ran~e of 0.47 - 8.8 x 105 daltons, and being labelled with tritium (3H) at the level of 100-1000 ~Ci/mg DNA. The 3H-DNA is diluted with stock solution of sample buffer~ which i5 made up of l.O ml of the above lBE buffer, 0.5 ml glycerol, 2.5 mg bromo-phenol blue, and 2.5 mg xylene cyanole FF. One volume of 3H-DNA
solution is mixed with one volume of sample buffer. The optimal total sample size is between- 10-30~. Each sample contains -from 0.3 ~Ci to 0.005 ~Ci of 3H-DNA which will separate into 11 bands of from 60,000 dpm/band to l,OOO dpm/band. The ~lycerol serves to make the *Trade Mark - 23 -3~

sample denser than the buffer for ease in application. The two dyes in the sample ~uffer serve as a visual clue to the progress of the ge1.
All of the gels were run at 100V for approximately 2 hours. The gels were dried for 1 to 2 hours on a "Bio-Rad"* brand dryer.
The autoradiography of the gels was conducted as follows:
An 8" x 10" sheet of KODAK* XAR-5 x-ray film was placed in a lead lined aluminum film cassette. The dried ge1s were placed on the film. The cassette was c10sed and clamped to insure good contact between the ge1s and the film. The cassette was wrapped in 2 1ayers 10 of aluminum foi1 to guarantee the 1ight sea1 and to prevent frost from forming on the cassette or film.
The cassette is placed in a -78''C freezer for the specified exposure time.
When the exposure is complete, the film cassette is removed frorn the freezer and warmed to room temperature (approximate1y 2 hours).
The fi1m is deve1oped as fo110ws:
KODAK X-RAY Developer 3 minutes NO AGITATION

STOP BATH (2% HAc)30 seconds CONSTANT
AGITATION

l(ODAK RAPID FIX5 minutes INTERMITTENT
AGITATION

Fluors Direct1y Incorporated in Po1y cry1amlde Ge1 A 5% gel with 10% Methoxypolyethyleneglycol 4-(2,5-diphenyl-oxa701yl) methy1 ether and 10% po1yethy1eneg1ycol di-1-naphthy1methyl ether was formed. 140 mg of the methoxypo1yethyleneglyco1 4-(2,5-di-phenyloxazo1yl) methyl ether of Examp1e 2 and 140 mg of the po1yethylene-glycol di-1-naphthy1methy1 ether of Examp1e 5 were disso1ved in 28 cc of acry1amide so1ution. The ge1 which was formed was uniformly impregnated with the fluorescent compounds. The 3H-DNA NET-644 (0.3 ,~Ci) was p1aced in each samp1e we11. One control gel was run.
30 It used no enhancement procedure. Two gels were placed on the same film. After 24 hours of exposure, the fi1m was not darkened by the ~¢,f *Trade Mark ~43~p~

control ael. The gel impregnated with the experimental fluors W25 clearly enhanced. Eleven, darkS discrete bands were visualized Post Electro _oresis Impregnation A 5~D polyacrylamide gel was prepared and run as described above with 0.15 uCi of Tritiated DNA (3H-DNA~ per sample. After electrophoresis was completed, the gel was soaked in a 5~ (w/v) aqueous solution of the methoxypolyethyleneglycol 4-(2,5-diphenyloxazolyl) methyl ether of Example 2.

This gel and a control gel were dried and exposed to film for 24 hrs. at -70C. The gel that had been soaked in the experimental fluor solution showed evidence of enhancement. Bands appeared in the expected pattern. No such pattern was produced by the control gel.

Post Electrophoresis A 5% acrylamide gel was prepared and run as described above with 50 ~Ci 3H-DNA per sample. After electrophoresis was completed, the gel was cut and the samples were soaked separately in 10mM and 20mM aqueous solutions of 4-[5-(2-phenyloxazolyl)3 benzene sulfonic acid (PP0-S03H).

The gels impregnated with the experimental fluor were clearly enhanced.
No such enhancement was observed by the control gel.
_AMPLE 20 A 5% acrylamide gel was prepared and run as described above with 50 ,uCi 3H-DNA per sample. After electrophores;s was completed, the gel was cut and soaked separately in lOOmM and 200mM aqueous solutions of naphthalene-2-sulfonic acid sodium salt. The gels impregnated with the experimental fluor were clearly enhanced.

A 5% acrylamide gel was prepared and run in the normal manner with 50 ~Ci 3H-DNA per sample. After electrophoresis was completed the gel was cut and soaked separately in the following aquecus solutions:

(A) 5mM PPO-S~3H and 200mM Naphthalene-2-sulfonic acid sodium salt (B) lOmM PPO-S03H and 100mM Naphthalene-2-sulfonic acid sodium salt.
(C) lOmM PPO-S03H and 200mM Naphthalene-~-sulfonic acid sodium salt.
(D) 10mM PPO-S03H and 300mM Naphthalene-2-sulfonic acid sodium salt.
(E) 20mM PPO-S03H and 200mM Naphthalene-2-sulfonic acid sodium salt.
(F) 20mM PPO-S03H and 300mM Naphthalene-2-sulfonic acid sodium salt.
(G) 20mM PPO-S03H and 400mM Naphthalene-2-sulfonic acid sodium salt.
The gels impregnated with experimental fluors were clearly enhanced.
_AMPLE 22 A 5~ acrylamide gel was prepared and run as described above with 50 ,uCi 3H-DNA per sample. After electrophoresis was completed, the gel was cut and the pieces soaked separately in lOmM, 20mM and 50mM 16% EtOH solutions of 2,5-diphenyl-3-methyloxa~olium toluene sulfonate.
The gels impregnated with the experimental fluor were clearly enhanced.

. _ A 2% agarose gel was prepared by dissolv-ing 1.0 gm of agarose in 50 ml of boiling buffer. The buffer consisted of 40mM Tris-HC1~
10mM sodium acetate, lmM EDTA (pH 7.5). The hot agarose solution is poured between two glass plates with a comb-shaped form to create sample wells. The gels are allowed to cool for several hours. The H-DNA was diluted with a sample buffer consisting of 80mM Tris-HCl, 20mM Na aceta~e, 2mM EDTA, 30% glycerol and 2.5 mg bromophenol blue, ., ~

3~

in a 1:1 ratio. The running buFfer was the same as the separating gel bufFer. The gels were electrophoresed at a constant 14mA. The electrophoresis was run as described above, with 100 ~iCi 3H-DNA per sample. After e1ectrophoresis was completed, the gel was cut and soaked separately in a solution having the composition of solution C
of Example 21.
The gels impregnated with experimental fluors were clearly enhanced.
While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made without departing from this invention in its broader aspects. Numerous other specific and unique advantages and applications of the present system will be readily apparent to those of skill in this art and are intended to come within the scope and spirit of the following claims.

Claims (48)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of enhancing the production of autoradio-graphic images by radioactive emitters contained in an absorbent or adsorbent separation medium, comprising con-tacting the separation medium with a fluorographic com-position, which composition comprises at least two water soluble fluors, each fluor having a structure in accordance with the formula.

wherein F is a component which absorbs energy and emits electromagnetic energy, S is a hydrophilic surfactant moiety which makes the fluor water soluble, and B is a chemical bond or other component which bonds a component F with a moiety S, and x, y and z are from 1 to 10.

2. The method of claim 1, wherein the fluorographic composition contains at least one water soluble fluor having a quantum efficiency above about 0.6.

3. The method of claim 1, wherein the fluorographic composition is mixed with the separation medium prior to inclusion of the radioactive emitters in said medium.

4. The method of claim 1, wherein the radioactive emitters are contained in the separation medium prior to contact with the fluorographic composition, and the fluoro-graphic composition is contacted with the separation medium in order to permit water soluble fluor contained in said fluorographic composition to transfer to the separation medium.

5. The method of claim 3 or 4, further comprising dry-ing the separation medium containing the water soluble fluors, and exposing a photosensitive film to the dried separation medium.

6. A method of enhancing the production of autoradio-graphic images from radioactive emitters containing an absorbent or adsorbent separation medium, comprising contact-ing the separation medium with a fluorographic composition, which composition comprises at least one water soluble fluor having a structure in accordance with the following formula:

wherein F is an organic radical which absorbs energy and emits electromagnetic energy, derived from 2,5-diphenyloxazole, 2,5-diphenyl-4-methyloxazole, naphthalene, l-methyl naphtha-lene, m-terphenyl, p-terphenyl, 3,3'-dimethyl-p-terphenyl, fluorene, 1,2-benzofluorene, l-methyl fluorene, isopropyl phenyl biphenylyloxadiazole, 2-[1-naphthyl]-5-phenyloxazole, t-butyl phenyl biphenylyl oxadiazole, S is a hydrophilic sur-factant moiety which renders the fluor water soluble, B is a chemical bond or other component which bonds a component F
with a moiety S, and x, y and z are integers from 1 to 10.

7. The method of claim 6, wherein S is a surfactant moiety derived from a polyethylene glycol, or a water soluble salt thereof, a primary, secondary or tertiary amine, a quaternary ammonium salt, an amide or a imide, or is a sulfonic acid radical or salt thereof.

8. The method of claim 7, wherein S is a surfactant moiety derived from a polyethylene glycol having a molecular weight of about 200 to about 1500, an aryl sulfonate, an amine, an amide, a quaternary ammonium salt, or S is a sul-fonic acid radical or salt thereof.

9. The method of claim 6, wherein B is a chemical bond or an alkylene, alkenyl, carboxy, arylene, alkarylene, or thio group.

10. The method of claim 9, wherein B is a chemical bond or an alkylene group having from 1-6 carbon atoms.

11. The method of claim 6, wherein the fluorographic composition is mixed with the separation medium prior to the inclusion of the radioactive emitters in said medium.

12. The method of claim 11, wherein the separation medium after admixture contains from about 0.01% to 85% by weight of water soluble fluor, based on the weight of the solids component of the separation medium prior to admixture.

13. The method of claim 6, wherein the radioactive emitters are contained in the separation medium prior to contact with the fluorographic composition, and the fluorographic composition is contacted with the separation medium in order to permit at least one water soluble fluor to transfer from the fluorographic composition into the separation medium.

14. The method of claim 13, wherein the contact is accomplished by immersion of the separation medium in the fluorographic compositions .

15. The method of claim 14 , wherein the separation material is contacted with the fluorographic composition for a period of less than two hours.

16. The method of claim 14, wherein the fluorographic composition contains water soluble fluor in an amount of from about 0.001M to about 2M.

17. The method of claim 13, wherein the fluorographic composition contains water soluble fluor in an amount of about 0.005M to about 1.0M.

18. The method of claim 13, wherein the fluorographic composition contains water soluble fluor in an amount of about 0.15M to 0.45M.

19. The method of claim 13, wherein the fluorographic composition contains the water soluble fluor methoxypolyethylene-glycol 4-(2,5-diphenyloxazolyl) methyl ether.

20. The method of claim 13, wherein the fluorographic composition contains 4-[5-(2-phenyloxazolyl)]benzene sulfonic acid or a soluble salt thereof in a concentration of about 0.005M to 1M and naphthalene-2-sulfonic acid or a soluble salt thereof in a concentration of about 0.005 to 1M.

21. A composition of matter, comprising at least two water soluble fluors, each fluor having a structure in accordance with the formula:

wherein F is a component which absorbs energy and emits electromagnetic energy, S is a hydrophilic surfactant moiety which makes the fluor water soluble, and B is a chemical bond or other component which bonds a component F with a moiety S, and x, y and z are from 1 to 10.

22. The composition of claim 21, wherein at least one water soluble fluor contained in the composition has a quantum efficiency of at least 0.6.

23. The composition of claim 21, wherein F is an organic radical derived from 2,5-diphenyloxazole, 2,5-diphenyl-4-methyloxazole, naphthalene, l-methyl naphthalene, m-terphenyl, p-terphenyl, 3,3'-dimethyl-p-terphenyl, fluorene, l,2-benzo-fluorene, l-methyl fluorene, isopropyl phenyl biphenylyloxa-diazole, 2-[l-naphthyl]-5-phenyloxaZOle, t-butylphenyl bi-phenylyl oxadiazole, S is a hydrophilic surfactant moiety which renders the fluor water soluble, B is a chemical bond or other component which bonds a component F with a moiety S, and x, y and z are integers from 1 to 10.

24. The composition of claim 23, wherein S is a hydro-philic surfactant moiety derived from a polyethylere glycol, sulfonic acid or a water soluble salt thereof, a primary, secondary or tertiary amine, a quaternary ammonium salt, an amide or an imide.

25. The composition of claim 24, wherein B is a chemical bond or an alkylene, alkenyl, carboxy, arylene, alkarylene or thio group.

26. The composition of claim 21, comprising an aqueous solution of 4-[5-(2-phenyloxazolyl)]benzene sulfonic acid or soluble salt thereof and naphthalene-2- sulfonic acid or a soluble salt thereof.

27. A composition of matter comprising a water soluble fluor having a quantum efficiency above about 0.6, and having a structure in accordance with the formula:

wherein F is a component which absorbs energy and emits electromagnetic energy, S is a hydrophilic surfactant moiety which makes the fluor water soluble, and B is a chemical bond or other component which bonds a component F with a moiety S, and x, y and z are from 1 to 10, and an acceptable carrier thereof.

28. The composition of claim 21 or 27, wherein each water soluble fluor has a quantum efficiency above about 0.1.

29. The composition of claim 21 or 27, wherein each water soluble fluor is present in an amount of from about 0.01% to about 85% by weight.

30. The composition of claim 21, wherein F is an organic radical derived from 2,5-diphenyloxazole, 2,5-diphenyl-4-methyloxazole, naphthalene, l-methyl naphthalene, m-terphenyl, p-terphenyl, 3,3'-dimethyl-p-terphenyl, fluorene, l,2-benzo-fluorene, l-methyl fluorene, isopropyl phenyl biphenylyloxa-diazole, 2-[l-naphthyl]-5-phenyloxazole, t-butyl phenyl bi-phenylyl oxadiazole, S is a surfactant radical which renders the fluor water soluble, B is a chemical bond or other com-ponent which bonds a component F with a radical S, and x, y and z are integers from 1 to 10.

31. The composition of claim 30, wherein S is a sur-factant radical derived from polyethylene glycol, or a water soluble salt thereof, from a primary, secondary or tertiary amine, a quaternary ammonium salt, from an amide or from an imide, or is a sulfonic acid radical.

32. The composition of claim 30, wherein S is a sur-factant radical derived from a polyethylene glycol having a molecular weight of about 200 to about 1500, an aryl sulfonate, an amine, an amide, a quarternary ammonium salt, or S is a sulfonic acid radical or salt thereof.

33. The composition of claim 31, wherein B is a chemical bond or an alkylene, alkenyl, carboxy, arylene, alkarylene or thio group.

34. The compositions of claim 21 or 27, wherein each water soluble fluor is selected from the group of 4-[5-(2-phenyloxazoyl)]benzene sulfonic acid or water soluble salt thereof, naphthalene-2-sulfonic acid or water soluble salt thereof, methoxypolyethyleneglycol 4-(2,5-diphenyloxolyl) methyl ether, polyethyleneglycol di-l-naphthylmethyl ether, 2,5-diphenyl-3-methyloxazolium toluene sulfonate, N-[4-(2,5-diphenyloxazolyl)methyl] acrylamide, N-[l-naphthylmethyl]-acrylamide, N-[3-(2,5-diphenyloxazolyl)methyl]-trimethyl ammonium chloride, N-[4-(2,5-diphenyloxazolyl) methyl]-N-benzyl-N,N-dimethyl ammonium chloride.

35. The composition of claim 21 or 27, wherein the water soluble fluor comprises a terpolymer of N-[4-(2,5-diphenyloxazolyl)methyl] acrylamide, N-[l-naphthylmethyl]-acrylamide and N-hydroxymethyl acrylamide.

36. The composition of claim 21, wherein at least one of said water soluble fluors fluoresces at a first wave-length, and at least one other fluor absorbs energy near said first wavelength and fluoresces at a second, desired wavelength.

37. The composition of matter of claim 21 or 23, wherein the composition in response to radiation, fluoresces at a wavelength which corresponds to the sensitivity of X-ray film.

38. An article of manufacture, comprising an absorbent or adsorbent separation medium containing the composition of claim 21.

39. An article of manufacture, comprising an absorbent or adsorbent separation medium containing the composition of claim 27.

40. The article of claim 38 or 39, wherein the separation medium is an electrophoresis gel.

41. The article of claim 38 or 39, further comprising a layer of photographic film attached to said separation medium, which film is sensitive to electromagnetic energy at a wavelength corresponding to the emission wavelength of at least one fluor contained in the separation medium.

42. The article of claim 38 or 39, further comprising radioactive emitters absorbed or adsorbed on the separation medium.

43. A method of enhancing the production of autoradio-graphic images by radioactive emitters contained in an absorbent or adsorbent separation medium, comprising con-tacting the separation medium with a fluorographic com-position, comprising at least one water soluble fluorescent material having a structure in accordance with the formula:

wherein F is a fluor moiety which absorbs energy and emits electromagnetic energy, S is a hydrophilic surfactant moiety which makes the fluorescent material soluble, and B is a chemical bond or other component which bonds moiety F with moiety S, and x, y and z are from 1 to 10.

44. The method of claim 43 wherein the fluorescent material has a quantum efficiency above about 0.6.

45. The method of claim 43 or 44, wherein the fluorographic composition is mixed with the separation medium prior to inclusion of the radioactive emitters in said medium.

46. The method of claim 43 or 44, wherein the radioactive emitters are contained in the separation medium prior to contact with the fluorographic composition, and the fluorographic composi-tion is contacted with the separation medium in order to permit water soluble fluor contained in said fluorographic composition to transfer to the separation medium.

47. The method of claim 43 or 44, wherein the fluorographic composition is mixed with the separation medium prior to inclusion of the radioactive emitters in said medium, and further comprising drying the separation medium containing the water soluble fluors, and exposing a photosensitive film to the dried separation medium.

48. The method of claim 43 or 44, wherein the radioactive emitters are contained in the separation medium prior to contact with the fluorographic composition, and the fluorographic composi-tion is contacted with the separation medium in order to permit water soluble fluor contained in said fluorographic composition to transfer to the separation medium, and further comprising drying the separation medium containing the water soluble fluors, and exposing a photosensitive film to the dried separation medium.